cyclic-gmp and diphenyliodonium

The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 micromol/L DPI under an atmosphere of 21% O(2) (5% CO(2)). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N(2) (5% CO(2)). If BCAs are treated with DPI under 21% O(2) and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N(2), there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O(2) but not under N(2). Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 micromol/L DPI under 21% O(2), but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O(2)-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.

Topics: Animals; Biphenyl Compounds; Cattle; Colforsin; Coronary Vessels; Cyclic GMP; Flavoproteins; Guanylate Cyclase; Hemeproteins; Muscle Relaxation; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Onium Compounds; Oxidation-Reduction; Oxidoreductases; Oxygen; Sulfates

The exposure of rat mesangial cells to cytokines promoted activation of the p42/p44 mitogen-activated protein kinase (MAPK). We identified a rapid and delayed phase of MAPK activation with distinctive activity increases at 5 to 15 min and 15 to 24 h. Rapid and late MAPK activation were attenuated by the redox-modulating agent N-acetylcysteine. Specifically, late-phase activation coincided with endogenous nitric oxide (NO) generation and in turn was suppressed by the NO synthase-blocking compounds diphenyliodonium or nitroarginine methyl ester. By using NO-liberating agents such as S-nitrosoglutathione and 3-morpholinosydnonimine, we investigated intermediary signaling elements of NO in promoting MAPK activation. Early and transient activation at 5 min was suppressed by the soluble guanylyl cyclase-blocking agent 1H-(1,2,4)-oxdiazolo-(4,3-alpha)-6-bromoquinoxazin-1-one (NS 2028) and, moreover, was mimicked by the lipophilic cyclic GMP (cGMP) analogue 8-bromo-cGMP. In contrast, NO-mediated activation achieved within hours was unrelated to cGMP signaling. Late and persistent MAPK activation, induced by NO donors or endogenously generated NO, was found in association with inhibition of phosphatase activity. In vitro dephosphorylation of activated and immunoprecipitated p42/p44 by cytosolic phosphatases was sensitive to the readdition of NO and was found to be inhibited in cytosol of S-nitrosoglutathione-stimulated cells. Also, cells that had been exposed to cytokines for 24 h revealed a blocked phosphatase activity, which was successfully attenuated by the NO synthase inhibitor nitroarginine methyl ester and, therefore, was NO mediated. Conclusively, NO affects p42/p44 MAPK in rat mesangial cells twofold: rapid activation is cGMP mediated, whereas late activation is transmitted via inhibition of tyrosine dephosphorylation.

Topics: Acetylcysteine; Animals; Biphenyl Compounds; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Cyclic GMP; Enzyme Activation; Free Radical Scavengers; Glomerular Mesangium; Glutathione; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Molsidomine; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Onium Compounds; Oxadiazoles; Oxazines; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Rats; S-Nitrosoglutathione; Second Messenger Systems

Diphenylene iodonium is an inhibitor of the enzyme NADPH-oxidase and prevents the generation of oxygen-derived free radicals in neutrophils (Cross and Jones, 1986). Here we show that diphenylene iodonium (0.25-2 microM) inhibited, according to the dose, thrombin-induced platelet-aggregation in human washed platelets and ADP-induced platelet aggregation in platelet-rich plasma. At the concentrations which inhibited platelet aggregation diphenylene iodonium did not alter platelet concentrations of cAMP or cGMP but enhanced the anti-platelet activity of iloprost, sodium nitroprusside or cultured endothelial cells. These findings highlight the importance of free radicals as platelet pro-aggregatory agents.

Topics: Adenosine Diphosphate; Biphenyl Compounds; Cyclic AMP; Cyclic GMP; Endothelium, Vascular; Free Radicals; Humans; Iloprost; In Vitro Techniques; Leukocytes; NADH, NADPH Oxidoreductases; Nitroprusside; Onium Compounds; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin